Natural and non-natural α-hydroxy-β-amino acids and β-hydroxy-γ-amino acids and their derivatives occur in many biologically active natural products and are important intermediates in the synthesis of various pharmaceuticals. One of the most important α-hydroxy-β-amino acids is the side chain of the potent anticancer drug Taxol. Various derivatives of this β-amino acid have been synthesized and linked to the polycyclic core ring of Taxol in an effort to improve the potency and the spectrum of uses of this important drug.
The β-hydroxy-γ-amino acid structural motif is encountered in a number of natural products and current and developmental drugs. Some of the most common β-hydroxy-γ-amino acids include statine, isostatine and benzyl statine (phenylstatine) (FIG. 1). Statine is the key component of pepstatin, a naturally-occurring hexapeptide antibiotic, which acts as an inhibitor of aspartic acid proteases such as rennin, pepsin and cathepsin D [Umezawa, H et al J. Antibiotics 23, 259 (1970); Ric, D. H. J. Med. Chem 23, 27 (1980)]. The low selectivity of pepstatine has led to the development of more specific synthetic analogues by substituting the isobutyl moiety of statine with more lipophilic substituents such as cyclohexylmethyl, which led to the widely used analogue cyclohexyl-statine. Isostatine is an essential amino acid in Didemnins [Sakai, R. at al J. Am. Chem. Soc. 117, 3734 (1995); Joullie, M. M. J. Am. Chem. Soc 112, 7659 (1990)), a group of cyclic peptides which show strong antitumor, antiviral, and immunosuppressive activity (Sakai, R. et al. J. Med. Chem. 39, 2819 (1996)]. Benzyl statine is part of the biologically active compounds hapalosin (Stratmann, K et al J. Org. Chem. 59, 7219 (1994); Armstrong, R. W. J. Org. Chem. 60, 8118, (1995)] and dolastatin 10 [Shiori, T et al Tetrahedron 49, 1913 (1993)]. In particular hapalosin restores the lethal activity of cytotoxic antitumor drugs (such as actinomycin D, colchicines and taxol) to cancer cells by breaking the β-glycoprotein-mediated multi-drug resistance caused by the export of the cancer drugs from the cell using transmembrane P-glycoproteins.





Figure 1: Natural products and related pharmaceuticals that contain γ-amino-β-hydroxy amino acids.
Other β-hydroxy-γ-amino acids that are incorporated in molecules with biological activities include (2S,3S,4R)-4-amino-3-hydroxy-2-methyl pentanoic acid, which is the amino acid linker of bleomycin B2 and the main constituent of the powerful carcinostatic blenoxane [Boger, D. L et al J. Am Chem Soc 116, 5607, (1994)] and (2R,3S,4R)-4-amino-3-hydroxy-2-methyl-5-(2′-pyridil) pentanoic acid, which is part of pyridomycin [Kinoshita, M; Awamura, M. Bull. Chem. Soc 51, 869 (1978)], a Streptomyces-synthesized anti-mycobacterial drug (Figure 2). Statines and related compounds based on β-hydroxy-γ-amino acids are particularly prevalent in anti-cancer drugs and drug candidates. The absolute stereochemistry of these molecules is important for biological activity.

b Figure 2: Potent biologically-active natural products that contain γ-amino- β-hydroxy amino acids.
Another important motif in pharmaceutically-active compounds is the α-hydroxy-β-amino acid structural unit. Among the examples of pharmaceutical products that contain the α-hydroxy-β-amino acid moiety as a key component in their structures are molecules such as bestatin, amastatin and ubenimex, which possess immunoregulatory, antitumor and antimicrobial activities. The ability to prepare compounds in this class with defined absolute stereochemistry is critical to the commercial synthesis of these compounds and their analogs.
Despite the general importance of hydroxyl-substituted β- and γ-amino acids and their derivatives as pharmaceutical intermediates, the preparation of these compounds remains a significant challenge to chemists. Most of the synthetic approaches toward the production of α-hydroxy-β-amino acids are purely chemical transformations that require multi-step reaction sequences, chiral catalysts or starting materials, and stringent or air-sensitive reaction conditions. Occasionally the synthetic methods involve the production of relatively unstable intermediates. Most of the chemical syntheses of statine and isostatine, for example, begin from the natural α-amino acids leucine and isoleucine, respectively [Hamada, Y. et al J. Am Chem Soc, 111, 669 (1989); Tao, J.; Hoffmann, R. V J. Org. Chem 62, 2292 (1997)]. After protection of the amino group (PG=protecting group), an aldol or Claisen condensation to the β-keto-γ-amino acid followed by a reduction gives the desired β-hydroxy γ-amino acid product (FIG. 3).
Figure 3: Outline of the most common current chemical syntheses of β-hydroxy-γ-amino acids.
Some of the problems encountered in these syntheses are the isomerization of the γ-carbon under the basic conditions of the condensation reaction, the many steps required (often 7–10), and the low diastereoselectivity of the final reduction step, which often times gives the wrong diastereomer as the major product [Kessler, H; Schudok, M Synthesis 457 (1990); Maibaum, J.; Rich, D. H J. Org. Chem 53, 869 (1988)]. An obvious drawback in using methods based on natural amino acid precursors for the synthesis of β-hydroxy-γ-amino acids is that non-natural α-amino acid counterparts cannot always be easily accessed, and for this reason other chemical synthetic schemes have been developed. The β,γ-amino alcohol moiety in one alternative synthetic route is synthesized from α,β-unsaturated alcohols that are epoxidized using a chiral catalyst, followed by a ring opening using an nitrogen nucleophile (FIG. 3) [Catasus, M. et al Tetrahedron Lett 40, 9309 (1999); Catejon, P. et al Tetrahedron 52 7063. (1996)]. Although good enantiomeric purity of the product was reported (90–99% ee), this methodology is long (6–10 steps), gives moderate yields (20–40%), and requires expensive catalysts and stringent air-sensitive reaction conditions. Other methods for synthesizing β-hydroxy-γ-amino acids involve Wittig reactions of chiral oxazolidinones [Reddy, G. V et al Tetrahedron Lett 40, 775 (1999)] asymmetric Claisen rearrangements [Krebs, A.; Kazmaier, U. Tetrahedron Lett. 40, 479 (1999)], selective Grignard reaction of N-protected amino acids [Veeresha, G.; Datta, A Tetrahedron Lett 38, 5223 (1997)] or the use of doubly chiral precursors [Kwon and Ko, Tetrahedron Lett 43, 639–641 (2002)]. Again, long and complicated reaction sequences and chiral starting materials and/or catalysts are required using these methodologies.
Enzyme catalysis offers an alternative to purely chemical synthetic schemes. Enzymatic methods that have been reported to date are resolutions of a racemic mixture, having a maximum yield of 50% for the resolution step alone. Challenges similar to those encountered in the chemical synthesis of β-hydroxy-γ-amino acids are also faced in the chemical synthesis of α-hydroxy-γ-amino acids. In both cases, gaining control over the stereochemistry of the chiral carbons bearing both the amino and the alcohol groups at reasonable cost and high enantiomeric purity is the key to the successful production of these important chemical intermediates.
Chiral hydroxy compounds can be produced by the stereoselective reduction of ketones catalyzed by ketoreductase enzymes. As used herein, the term ketoreductase means any enzyme that catalyzes the reduction of a ketone to form the corresponding alcohol. Ketoreductase enzymes include those classified under the Enzyme Commission numbers of 1.1.1. Such enzymes are given various names in addition to ketoreductase, including, but not limited, to alcohol dehydrogenase, carbonyl reductase, lactate dehydrogenase, hydroxyacid dehydrogenase, hydroxyisocaproate dehydrogenase, β-hydroxybutyrate dehydrogenase, steroid dehydrogenase, sorbitol dehydrogenase, aldoreductase, and the like.
Many examples of enzymatic reductions of various classes of substrates have been reported [Wong, C-H; Whitsides, G. M. Enzymes in Synthetic Organic Chemistry, Pergamon, New York, (1994); Sugai, T Curr. Org. Chem 3, 373 (1999)]. Various alcohol dehydrogenases have been investigated [Patel, R. N Adv. Appl. Microbiol 43, 91 (1997); Riva, S.; Carrea, G. Angew. Chem. Int. Ed 39, 2226 (2000)]. A well known example is horse liver alcohol dehydrogenase (HLADH), an enzyme that has been very extensively studied and can reduce aldehydes and ketones to the corresponding alcohols, in some cases providing alcohols in good enantiomeric purity. The substrate range is limited and does not include most β-ketoesters, however.
Various ketoreductase enzymes have been identified that catalyze the stereoselective reduction of a range of different ketones, including β-ketoesters. [See, for example, J. David Rozzell, ACS Symposium Series 776, Applied Biocatalysis in Specialty Chemicals and Pharmaceuticals, B. C. Saha and D. C. Demirjian, eds., pp.191–199, (2000) and references therein, all hereby incorporated by reference.] These enzymes have been shown to act on a number of structurally diverse ketones. The genes expressing a number of these broad-range ketoreductases have been cloned and expressed, and a number of these enzymes are readily available commercially (BioCatalytics, Inc, Pasadena, Calif. USA). In many cases, enzymes can be identified that can produce either stereoisomer of a chiral alcohol by stereoselective reduction of a target ketone. For example, when the Ketoreductase Screening Set (Catalog number KRED-8000, BioCatalytics, Inc, Pasadena, Calif. USA) containing 8 different ketoreductases was screened against either alpha-chloroacetophenone or ethyl 4-chloroacetoacetate, some enzymes could be found within the set that were R-selective while others were found that were S-selective with respect to the chiral alcohol produced.
It has also been demonstrated that ketoreductase enzymes can be used to catalyze the reduction of 2-substituted-3-ketoesters. The products of these reductions are compounds with two chiral centers, and depending on the enzyme employed, the reduction can be diastereoselective, as shown in FIG. 4. Such reactions have been described using isolated enzymes and with whole cells. When the enzymes within the Ketoreductase Screening Set (Catalog number KRED-8000, BioCatalytics, Inc, Pasadena, Calif. USA) were studied for the reduction of 2-ethyl-3-ketobutyrate ethyl ester, certain enzymes were shown to be highly diastereoselective for the reduction to the corresponding alcohol. [For other examples, see S. Rodriguez et al., J. Org. Chem., 65, 2586 (2000); S. Rodriguez et al., J. Am. Chem. Soc., 123, 1547 (2001) and references therein, hereby incorporated by reference.]
Figure 4: Diastereoselective reduction of 2- substituted-3-ketoesters
In contrast to the 2-substituted-3-ketoesters shown in FIG. 4, there is only a single report of the diastereoselective reduction of a β-ketodiester such as that depicted in FIG. 5.
Figure 5: Diastereoselective reduction of β-ketodiesters
Benner and coworkers used actively fermenting Baker's yeast to carry out the reduction of the compounds shown in FIG. 5 where n is 1 and R is allyl or propargyl [T. Arsian and S.
A. Benner, J. Org. Chem., 58, 2260–2264 (1993) and references therein, hereby incorporated by reference]. These compounds were prepared as potential precursors for the synthesis of non-standard nucleic acid bases. These were the only compounds for which reduction with fermenting yeast was reported, and the ketoreductase or ketoreductases involved were neither isolated nor determined. The reduction reaction was reported to be enantioselective and diastereoselective, although the degree of selectivity observed varied widely depending on reaction conditions, and yields in some cases were diminished by the partial metabolism of the substrate.
There are no reports of the highly diastereoselective reduction of a range of substituted β-ketodiesters, nor any reports of the use of substituted β-ketodiesters in the production of α-hydroxy-β-amino acids and β-hydroxy-γ-amino acids using a reaction sequence incorporating a diasereoselective reduction of substituted β-ketodiesters.